Cytokines

More than 13 chemokine receptors are associated with rheumatoid arthritis, and at least 16 chemokines interact with these receptors.⁹ Both constitutive (responsible for physiologic trafficking and homing of the adaptive immune response) and inducible (responsible for effector white blood cell recruitment, including lymphocytes) responses are involved. A number of the chemicals also are responsible for angiogenesis. The inappropriate production of chemokines is associated with formation of an ectopic germinal center, which contributes to an uncontrolled immune response. Consequently, drugs directed toward chemokine receptors offer a therapeutic approach to control.

TNFα induces a broad spectrum of activity. Cytotoxicity of multiple cell types, including tumors, reflects both apoptosis and necrosis. A large number of proteins are targeted, along with other central mediators of the inflammatory process and immune activation. Examples include nuclear factors (e.g., NFκB), nitric oxide synthetase (NOS), cell-surface molecules, MHC classes I and II, and secreted proteins such as IL (e.g., -l, -6, -8), IFN, granulocyte-macrophage colony-stimulating factor, platelet-derived growth factor, urokinase plasminogen activator, and TNF-α itself. When administered exogenously, high concentrations of TNFα causes a toxic syndrome similar to septic shock. Bacterial lipopolysaccharide is the most potent stimulator of macrophage TNFα production. TNFα appears to mediate ischemia–reperfusion injury after transplantation of the liver, kidney, intestine, heart, lung, and pancreas and is a marker cytokine during organ rejection. Inhibitors have proved useful in human medicine for treatment of a number of autoimmune diseases, including Crohn’s disease, rheumatoid arthritis, psoriasis, and ankylosing spondylitis. Indications included corticosteroid-resistant graft-versus-host disease after bone marrow transplantation.¹⁰

Not surprisingly, the pervasive role of TNFα has led to the development of drugs that inhibit its actions. Included are glucocorticoids, pentoxifylline, and monoclonal immunoglobulin G (IgG) antibodies (e.g, infliximab, etanercept and the “humanized” adalimumab) etanercept, a humanized soluble TNFα receptor (TNFR) construct, and onercept, a TNFα–binding protein.¹⁰ A variety of other drugs targeting TNFα or its receptors are currently under investigation, as well as metalloproteinase inhibitors such as TNFα-converting enzyme (TACE). The use of these drugs, not surprisingly, is associated with a number of side effects. These include immunosuppresion, acute infusion reactions, delayed-type hypersensitivity reactions, autoimmune diseases, cardiovascular and neurologic adverse events, malignancies and lymphomas, and infectious complications.

Complement

The complement cascade is a major effector mechanism of the immune response. The cascade results in highly amplified events that interact with other physiologic cascades, including the coagulation pathway, kinin formation, and fibrinolysis.¹¹ Consequences of complement activation include opsonization, the release of biologically vasoactive peptides, and cellular lysis. Cell-mediated cytotoxicity is implemented by cytotoxic T lymphocytes, NK cells and antibody-dependent cell-mediated cytotoxicity (ADCC), mediated by a variety of cells that express surface Fc receptors. Effector cells of ADCC include monocytes, neutrophils, eosinophils, selected cytotoxic T cells, and NK cells.

Immunoglobulins

Five classes of Igs are recognized in animals. IgM is the largest, forming up to 15% of the total Ig present. IgM exists as a monomer or a large polymeric form. IgM is responsible for the primary Ig response; in animals, for some infections, it is the only defense.¹¹ The availability of five binding sites renders IgM efficient at antigen binding and agglutination, virus neutralization, and opsonization. IgM also is a potent activator of complement. Because it is such a large molecule, unless vascular permeability is altered, most IgM stays in circulation.

IgG (with multiple subclasses) makes up the majority of total Ig. It is the most soluble of the Igs and thus is able to reach extravascular spaces. Its primary biological functions are to facilitate the removal of microorganisms; neutralize toxins; and bind to microorganisms or infected cells, initiating effector mechanisms. IgG activates complement or initiates Fc-bearing effector cells (ADCC) and promotes removal of Ig-coated cells by ADCC.

IgE has a major role in the response to parasites and in the pathogenesis of allergic diseases in part because of its unique ability to bind by way of the Fc portion of the IgE molecule to specific receptors on mast cells and basophils. Cross-linkage of two IgE molecules results in calcium-mediated mast cell degranulation and the release of a number of preformed (e.g., histamine and serotonin) and synthesized (e.g., metabolites of arachidonic acid) mediators.

IgA is produced in submucosal lymphoid tissues and regional lymph nodes. After secretion outside of the cell, it travels to epithelial cells, where the secretory component of the IgA acts as a receptor, binding to IgA and stimulating its endocytosis. The two components are eventually exocytosed and attach to the mucosal surface, where they provide a protective component, neutralize toxins, adhere to bacteria and viruses, and interact with parasites.¹¹

Each Ig molecule monomer comprises two heavy chains and two light chains attached by covalent bonds. The number of bonds varies with the Ig; the number of dimers varies with the class. A number of fragments (generated by enzymatic cleavage) can be described, including Fab, the antigen-combining fragment, and Fc, the crystallizable fragment, which binds to Fc receptors found on cells of the innatue immune system (e.g., NK cells, macrophages, neutrophils and mast cells).¹¹

Calcineurin Inhibitors

Calcineurin is a protein phosphatase that dephosphorylates and thus activates the transcription factor nuclear factor of activated T cell (NFATc). Upon activation, NFATc translocates to the nucleus and upregulates IL-2 expression of regulatory proteins.

Chemistry–Structure Relationship

Cyclosporin A (CsA), now known as cyclosporine, is the most important immunosuppressive drug for human transplantation and the treatment of selective autoimmune disorders.^4,30 CsA is one of nine cyclosporins (A through I), each a cyclic peptide drug (Figure 31-3) isolated from the fungi Cylindrocarpon lucidum and Trichoderma polysporum. An M designation, when present, indicates a metabolite. The drug is both very lipophilic (hydrophobic) and must be solubilized before administration.⁴ The oral preparation is a soft gelatin capsule (Sandimmune) or a (newer) microemulsion formulation (Neoral). The intravenous preparation is an ethanol–polyoxyethylated castor oil mixture. Historically, CsA has been formulated in peanut oil (for treatment of ophthalmic ocular disorders in dogs); however, an approved product formulated for topical use is now available.

Figure 31-3 The chemical structure of selected immunomodulating drugs.

Mechanism of Action

CsA is a unique immunosuppressant in that it specifically inhibits T_h cells (both T_h1 and T_h2) early in their immune response to antigenic and regulatory stimuli⁴ without affecting suppressor cells. Suppression occurs as CsA binds to cyclophilin, a cytoplasmic receptor protein, forming a heterodimeric complex. This complex then binds to calcineurin. Binding of calcineurin inhibits calcium-stimulated phosphatase that dephosphorylates the regulatory protein. As such, CsA prevents transcription of T cell genes enhanced in response to T cell activation. Transcription mediated by IL-2, certain proto-oncogenes, and selected cytokine receptors are particularly affected.⁴ IL-2 production (and thus T cell proliferation and antigen-specific cytotoxic T lymphocyte generation) also is attenuated because the expression of TGF-β, a potent inhibitor of IL-2, is increased.⁴ B cells are not affected. The drug is most effective when administered before T cell proliferation has occurred. In addition to these immunomodulatory effects, CsA also affects other inflammatory cells. CsA inhibits skin mast cell numbers, survival, and response (secretory and histamine release) as well as secretion of IL-3, IL-4, IL-5, IL-8, and TNFα. Similarly, eosinophile response (release of granules and cytokines) and recruitment to allergic sites is impaired. CsA prevents TNFα-mediated late phase reactions, thus inhibiting IgE and mast cell–dependent cellular infiltration in the skin and bronchial mucosa. However, CsA does not inhibit IgA secretion or IgG and IgM formation in dogs and has no apparent effect on serum allergen-specific IgE levels, intradermal tests, or vaccination.

Surprisingly little information is available regarding immunomodulatory effects of CsA in dogs or cats. Using dermal microdialysis, Brazis and coworkers³¹ demonstrated that CsA at 5 mg/kg per day for 15 and 30 days decreased histamine, but not prostaglandin D₂, release in sensitized Beagles challenged with Ascaris suum.

Preparations

CsA is available in a variety of preparations, two of which are approved in the United States for use in animals. CsA was first formulated for oral use in humans as vegetable oil. Oral absorption in this preparation depends on emulsification by bile acids. Poor oral bioavailability led to the formulation of a microemulsion preparation (modified CsA, ME), which, on contact with gastrointestinal fluids, disperses into a homogeneous monophasic microemulsion that mimics the mixed micellar phase of the standard formulation. In dogs the ME formulation offers a 35% bioavailability (Novartis Animal Health data on file).

Atopica is the veterinary-approved microemulstion preparation of CsA intended for systemic use in both capsules and solutions. A 0.2% ophthalmic ointment (Optimmune) is also available. A number of human-approved drugs are available, including an injectable solution, an oral solution (100 mg/mL), an ophthalmic emulsion (0.05%), and a variety of capsule sizes for the standard and the microemulsion-modified preparation. Included are a number of generic-modified CsA products. However, the therapeutic equivalence that has been established for these products in humans should not be assumed to be therapeutically equivalent to Atopica in animals. Indeed, one preparation made by IVAX Pharmaceuticals and sold by selected chain pharmacies appears to result in much higher concentrations in dogs compared with other preparations on the basis of samples submitted through the author’s laboratory. Monitoring should be the basis for assessing the appropriate dose–concentration relationship for oral human CsA products used in dogs or cats; owners might need to be aware if and when a pharmacy switches from one human generic to another such that prophylactic monitoring might be implemented.

Clinical Pharmacology

The pharmacokinetic behavior of CsA is complex, resulting in marked variability in the relationship between dose and blood concentrations.³² Variability reflects not only differences in both hepatic and intestinal metabolism (by CYP3A4) but also differences in P-glycoprotein among various tissues.³² The disposition of CsA has been well reviewed in the dog.³³ Absorption after oral administration is slow and incomplete. The complex lipid nature of CsA complicates absorption by being dependent on bile acids, which generate a microemulsion. In humans oral bioavailability of the capsule ranges from 20% to 50% but is improved by 10% to 20% in the microemulsion form which is not dependent on bile acids.^4,34 Peak concentrations occur 1 to 4 hours after oral administration in human beings. When the capsule—but not the microemulsion—is administered with a fatty meal, absorption is slowed. Studies suggest that decreased bioavailability of CsA after oral administration may reflect activity of drug-metabolizing enzymes of the intestinal epithelium³⁵ or P-glycoprotein–mediated drug efflux.

The oral bioavailability of microemulsion in dogs is 35% compared with the 20% of the oral preparation.³³ Bioavailability of orally administered CsA is approximately 25% to 30% after multiple oral administration in cats (n = 6). Food may impair the absorption of CsA; peak concentrations in fasted dogs were 20% higher than in nonfasted dogs in one study, although time to peak (approximately 1.5 hours) was the same.³³ The drug is not predictably absorbed topically after transdermal administration (unpublished data, Boothe 2007). CsA is widely distributed, characterized by a large volume of distribution (13 L/kg in humans). The drug accumulates in erythrocytes (accounting for 50% or more of the drug in humans), and leukocytes (accounting for 10% to 20% of circulating drug in humans).⁴ Consequently, monitoring of whole blood rather than plasma or serum is recommended. Remaining circulating drug is bound to plasma lipoproteins. Further, concentrations in skin are up to tenfold higher than in blood, although this is based on homogenate data (as reviewed by Guaguère and coworkers³³). In contrast, largely because of the influence of adenosine triphosphate–binding transporters P-glycoproteins, especially the ABCB1 cassette (P-glycoprotein, MDR1 gene product), little CsA crosses the blood–brain barrier.

CsA is metabolized to a large number of metabolites predominantly by the liver, although sufficient metabolism occurs by intestinal enterocytes that oral bioavailability is affected. Microflorae also may contribute to metabolism.³⁷ CYP3A4 (commonly associated with P-glycoprotein or other efflux pumps) plays a major role in metabolism; many of the drug interactions involving CsA also involve CYP3A4 or associated glycoprotein. More than 25 to 30 metabolites have been documented in humans and dogs.^32,38,39 Metabolism is targeted primarily toward the side chains rather than the ring structure and reflects hydroxylation, demethylation, sulfation, and cyclization. The major hydroxylated metabolites (AM1 and AM9) are further metabolized, contributing to the complex metabolic profile. Both AM1 and AM9 may contribute 10 to 20% of CsA activity, with AM1 contributing 27% of trough activity.⁴⁰ In humans AM1 accumulates with chronic dosing and ultimately may surpass CsA, and monitoring assays might ideally detect the active compounds.³⁹ They are detected to variable degrees by selected immunoassays based on a monoclonal antibody (e.g., Abbott TdX, Architect systems, Seimens Dimension).^41,42 Selected laboratories may also offer a high-performance liquid chromatography (HPLC)–based system that, may or may not (generally not) quantitate the metabolites.³⁹ Ultimately metabolism continues until the drug is totally inactivate the drug, although this has not been proved conclusively.⁴ Metabolites are excreted in bile and feces, with less than 2% of unchanged drug eliminated in the kidneys of dogs. CYP3A4 is largely responsible for metabolism.

The half-life of CsA in dogs is quite variable, depending on the investigator and assay. In blood the half-life ranges from a low of 5 hours (HPLC) to a high of 18 hours (using fluorescent polarized immunoassay [FPIA]); 29 hours was reported for serum.⁴³ That reported for Atopica, the product approved for use in dogs, is 4.5 hours, a length more consistent with that measured in the author’s therapeutic drug monitoring laboratory using FPIA. In a manufacturer-sponsored affinity colony-mediated immunoassay (ACMIA) study, Steffan and coworkers⁴⁴ reported the disposition of CsA (5 mg/kg) after single-dose oral administration of either capsules or solution in fasting or fed conditions using a randomized crossover design with a 1-week washout between studies (Table 31-3); food decreased peak concentrations and area under the curve by 22%.

Table 31-3 Disposition of Cyclosporine Reported By Various Investigators

In cats elimination half-life of CsA is 8 hours, which was similar to that after intravenous administration. Liver disease will decrease clearance of CsA substantially. However, for the nonmicroemulsion form, decreased oral absorption in the face of decreased bile may balance decreased clearance. Monitoring is recommended in the presence of hepatic or gastrointestinal disease.

Mehl and coworkers⁴⁵ reported the disposition of CsA in cats (n = 6 male) after single-dose intravenous (1 mg/kg) and multiple-dose (14 days; 3 mg/kg twice daily) as Neoral (see Table 31-3). Variability in plasma drug concentrations among cats and across time underlines the importance of monitoring if treating life-threatening conditions. After multiple-day (7 to 14 days) oral administration (3 mg/kg), mean (peak) plasma concentrations at 2 hours were 740 ± 326 (7 days) and 655 ± 285 ng/mL (14 days), whereas trough concentrations were 332 ± 237 and 301.5 ± 250 ng/mL, respectively. Oral bioavailability was only 25% to 29%.

Because cats may find the oral preparation unpalatable, the oral solution can be given diluted in olive oil. The intravenous preparation (4 to 6 mg/kg as a 4-hour intravenous infusion) can be given to animals for which oral administration is not possible.⁴⁶ Ocular administration might serve as an alternative route for systemic delivery of CsA in cats.⁴⁷ After ocular administration of either an oral preparation or an ocular preparation of CsA in olive oil, (5 mg/kg in each eye as a 10% solution) peak concentrations of 450 to 1033 ng/mL (oral preparation) and 288 to 648 ng/mL (ocular) were achieved, with an absorption lag time ranging from 0 to 1.34 hours for the oral solution and 0.27 to 1.2 hours for ocular preparation. The elimination half-life ranged from 2.41 to 10.04 hours (oral), and 3.09 to 15.75 hours (ocular), respectively. Blood concentrations were sufficient to inhibit lymphocyte activity as measured in vitro, leading the authors to conclude that ocular administration of CsA in an olive oil vehicle might be a reasonable alternative to oral, or even intravenous, administration in cats intolerant to the latter.

Drug Interactions

Novartis⁴⁸ provides a transplant drug interactions monograph that cites more than 600 references and delineates cyclosporine interactions involving more than 300 drugs. The list of drugs that might interact with CsA is extensive. The manual is available on the Novartis website at no charge and should be consulted when treating patients receiving additional drugs. Selected interactions can be found in Table 31-4. This review will focus on some of the more common interactions, including those used intentionally.

Table 31-4 Cyclosporine Drug Interactions

ACE, angiotensin-converting enzyme; CsA, cyclosporine A.

From Neoral and Sandimmune Drug Interactions, Novartis package insert, 2007

The enzyme that metabolizes CsA (CYP3A4) is responsible (in humans) for approximately 30% of all drug metabolism; many of CsA drug interactions occur with this enzyme. Further, CsA is a target of P-glycoprotein, another site characterized by a substantial number of CsA–drug interactions.^4,49 The risk of drug interactions is another indication of the need for therapeutic drug monitoring as a guide to proper dosing regimens. CsA elimination is accelerated, probably in part because of the induction of drug-metabolizing enzymes, by phenytoin, phenobarbital, rifampin, and sulfamethoxazole–trimethoprim combinations, and others. Dexamethasone also is an inducer of CsA.³² Its elimination is decreased by amphotericin B, erythromycin, and ketoconazole.⁴ CsA also appears to alter the oral absorption of other drugs.³⁵ CsA use with other immunosuppressive drugs may benefit from these interactions, including glucocorticoids.⁴

The inhibitory effect of ketoconazole on intestinal epithelial and hepatic drug metabolism and on P-glycoprotein efflux may be of therapeutic benefit in patients receiving CsA by decreasing hepatic clearance and increasing oral bioavailability.^35,50-53 Ketoconazole may also decrease the lipoprotein that binds CsA, resulting in higher concentrations of free CsA.⁵¹ Studies with ketoconazole in humans found the dose of CsA to be reduced by approximately 70% to 85%.⁵⁴ The use of ketoconazole in conjunction with CsA as a means of decreasing drug cost has been well accepted in human transplant patients.⁵¹ The two drugs have been used safely for up to 47 months in one study reducing the CsA dose as much as 88%.⁵¹ Although the effects emerge rapidly, with 62% of the effect is apparent by day 7, the maximum inhibitory effect may not be present for 12 months. This has implications regarding monitoring and supports the need for collection of peak and trough samples such that the impact of ketoconazole on drug elimination half-life might be determined.

The effect of combining ketoconazole with CsA has been studied in dogs.⁵⁵ Ketoconazole was studied at doses ranging from 1.25 to 20 mg/kg per day, with the magnitude of inhibition increasing with the dose of ketoconazole above but not below 2.5 mg/kg.⁵⁵ Clearance was reduced by 85% at 10 mg/kg per day. Differences in clearance did not result in significant differences in oral bioavailability. In their review, McNaulty and Lensmeyer⁵⁶ indicated that in dogs, CsA half-life will increase over twofold and decrease the concentration of active metabolites in response to 2.5 to 10 mg/kg ketoconazole. Up to 75% of the CsA dose could be reduced experimentally in Beagles concurrently receiving a dose of ketoconazole of 13.6 mg/kg per day.⁵⁷ In a study in dogs with perianal fistulae, Mouatt⁵⁸ examined the impact of ketoconazole (10 mg/kg once daily) on CsA concentrations (1 mg/kg twice daily). Samples were collected 12 hours after the last dose (duration not clear). Data were not provided on all dogs, but the author noted that at 1.1 mg/kg twice daily of CsA, all dogs exceeded 200 ng/mL at through (12 hours); nine dogs exceeded 900 ng/mL, and two dogs exceeded 1500 ng/mL. Concentrations varied by 10% to 40% in the same dog despite no dose change. This may have reflected variability in time to steady state, which took 2 to 4 weeks. A trough concentration of 200 ng/mL was targeted. Although 8 dogs maintained concentrations of 220 to 520 ng/mL with CsA doses as little as 0.35 to 0.55 mg/kg twice daily, one dog required only 0.16 mg/kg twice daily to maintain 159 to 225 ng/mL, whereas three others required 0.95 to 1.1 mg/kg twice daily to maintain 120 to 235 ng/mL. This marked variability in response to ketaconozole appears to occur in clinical patients as is suggested by samples submitted to the author’s therapeutic drug monitoring laboratory. Marked variability occurs not only in concentrations, but in the time to steady-state, which in turn determines when monitoring should be implemented. For example, the author’s laboratory has demonstrated prolongation of the half-life of CsA in a patient from 4 hours (as reported in normal dogs) to over 150 hour. Steady state was not achieved in this patient until approximately 4 weeks, causing CsA concentrations to continue to increase despite a decrease in dose that was based on monitoring at 1 week. Yet, in other patients, half-life has been less than 12 hours, despite ketoconazole therapy. Detecting these diffrences requires collection of peak and trough samples such that half-life and time to steady-state can be determined in each patient.

The simultaneous administration of ketoconazole with CsA also has been studied in cats. McNaulty and Lensmeyer⁵⁶ prospectively studied the impact of a two doses of ketoconazole (10 mg/kg at 24 and 0.5 hr) before a single dose of CsA (4 mg/kg intravenously) in cats (n = 5) using a randomized crossover design with a 14-day washout. Concentrations of CsA were detected using HPLC. Clearance was reduced from 2.73 ± 0.3 to 1.22 ± 0.18 mL^∗kg/min, resulting in an elimination half-life prolongation from 10 ± 0.9 to 21 ± 3 hours.

Several studies have examined the impact of cimetidine, an inhibitor of CYP3A4, CYP2D6, and CYP1A2 on CsA concentrations. Cimetidine also is a substrate for P-glycoprotein. The results of the studies are contradictory, perhaps reflecting differences in duration of therapy and species. D’Souza and coworkers⁵⁹ found that 5 days of cimetidine therapy significantly decreased CsA clearance, increased volume of distribution (perhaps by impacting P-glycoprotein in distribution tissues) and prolonged elimination half-life in rabbits, whereas Bar-Meir and coworkers⁶⁰ found that short-term administration of cimetidine had no effect on CsA metabolism in rats. Shaefer and coworkers⁶¹ found that 7 days of dosing with cimetidine or famotidine did not affect CsA pharmacokinetics in healthy men. Daigle⁶² explored the impact of cimetidine (15 mg/kg orally every 8 hours for 8 days) on the disposition of CsA (5 mg/kg orally per day; the last 3 days of cimetidine administration) in dogs using a randomized crossover design with a 14-day washout. Significant differences could not be demonstrated for C_max or area under the curve the power to detect a significant change was not addressed. The author’s therapeutic drug monitoring laboratory has identified some patients for which a cimetidine–CsA interaction may be present, but, other patients for which no effect could be measured.

Among the other drugs that impair CsA elimination or compete with efflux pumps include itraconazole, the calcium channel blockers such as diltiazem, and the macrolide antibacterial erythromycin.⁵⁰ In humans, diltiazem decreases the CsA dose by approximately 30% to 50%.⁵⁴ Other drugs that influence CYP activity should be expected to potentially affect CsA. For example, in the author’s laboratory, a cat receiving azithromycin (5 mg/kg once daily) along with CsA (5 mg/kg twice daily), exhibited peak concentrations exceeded 4500 ng/mL, presumably in part resulting from an elimination half-life that exceeded 150 hours. Azithromycin also competes for P-glycoprotein. Other drugs that induce P-glycoprotein might decrease CsA concentrations. For example, P-glycoprotein is upregulated in the duodenum of dogs receiving glucocorticoids.^62a

Side Effects

CsA is characterized by a narrow therapeutic index in human patients, with renal toxicity the primary adverse effect. Renal tubular cells develop hyperuricemia (worsened by diuretics) and hyperkalemia (a renal tubular and erythrocyte ion channel effect).⁶³ Hepatic injury also occurs. Although less common than renal dysfunction, the risk of severe hepatic damage is markedly increased when CsA is used in combination with cytotoxic drugs. CsA also increases the incidence of gallstones in human patients. However, in contrast to humans, renal and hepatic toxicities do not appear to be in dogs and cats. Risk factors, such as concurrent administration of nephroactive or nephrotoxic drugs, or renal transplantation have not been addressed in dogs or cats. Hyperlipidemia also has been a reported side effect in humans, particularly in patients receiving glucocorticoids. Other side effects reported in humans include neurotoxicity, gastrointestinal upset, and hypertension.⁴ Development of B cell lymphoma also has been reported in humans; an incidence 10% has been reported in cats undergoing renal transplantation, with mean time to onset of 9 months. ^63a Side effects in dogs include gastrointestinal upset and dermatologic or mucosal abnormalities. Vomiting may occur in up to 40% of dogs, although it may be intermittent and short in duration. Diarrhea occurs less commonly (16% to 18%).Vomiting may be more likely when CsA is combined with ketoconazole, but this may reflect higher CsA concentrations. Dermatologic abnormalities reported in dogs include hair loss (possibly reflecting hair growth and pushing of hair from follicles), gingival hyperplasia, and gingivitis in up to 33% of dogs.⁵⁸ In one study,⁵⁸ hair loss resolved by 7 weeks, although hairy coats were thinner at study end (see Therapeutic Use). Hyperplasia may reflect an imbalance of fibroblast formation and collagen degradation by collagenase, although stimulation of TGF-β also has been suggested.³³ Stimulation of TGF-β also stimulates extracellular matrix and decreases degrading proteases. Treatment with antimicrobials (metronidazole, spiramycin) may be useful for treating hyperplasia.⁵⁸ Skin healing might be supported rather than inhibited, as occurs with glucocorticoids.⁶⁷

At the doses used to treat atopy, risk of infection does not appear to be increased in dogs. However, the risk apparently has not been assessed at the higher doses used to treat immunosuppressive diseases. A report of central nervous system (CNS) toxoplasmosis has been reported in two cats receiving CsA (3 mg/kg twice daily for approximately 5 weeks and 6 mg/kg twice daily for approximately 8 weeks, respectively); presenting signs were respiratory in nature. Both had have previously been treated with glucocorticoids for a substantial time before diagnosis. A fatal case of toxoplasmosis was reported in a cat afflicted with eosinophilic granuloma complex receiving CsA at 5 mg/kg per day for a month, which was reduced because of anorexia to every other day. After 6 months of therapy, the patient developed fatal acute hepatic failure when the dose was increased to once daily. Toxoplasmosis was detected histologically in a number of tissues.⁶⁷ CsA apparently inhibits insulin secretion in a dose-dependent manner in humans.³³ Although this does not appear to be true in dogs, prudence dictates close monitoring of insulin needs in diabetic animals receiving CsA. ^63b In rare cases neurotoxocity, characterized by reversible cortical blindness, has been reported in human patients receiving CsA after bone marrow transplantation.⁶⁴

Mouatt⁵⁸ reported that 11 of 16 dogs receiving ketoconazole with CsA (Neoral solution) developed hair loss manifested as excessive shedding; five animals also developed metronidazole-responsive gingivitis with hyperplasia. Steffan⁶⁵ compared adverse events associated with CsA (n = 119; 5 mg/kg per day induction followed by tapering doses) versus methylprednisolone (n = 59; 0.5 to 1 mg/kg/day for 1 week, then every other day for 3 weeks, then tapered) in dogs with atopic dermatitis. The incidence of selected adverse events differed between groups. The most common in the CsA group were gastrointestinal, for a total of 55%. Among these, the most common was vomiting at 37%, which was significantly more than the methylprednisolone group (5%), followed by soft stools and diarrhea (18%). Infections occurred with similar frequency between groups but were more likely to be classified as severe or very severe in the methylprednisolone-treated group and led to more dropouts in that group. Polyuria, polydipsia, and increased appetite were more common in the methylprednisolone-treated group. Gingival hyperplasia was reported in 3% of CsA patients (none in the methylprednisolone-treated group). Serum chemistries indicative of renal function did not change from baseline. Potassium increased by approximately 0.6 mg/dL in the CsA-treated group (and decreased in the methylprednisolone-treated group) but was within normal values.

In their meta-anlysis of clinical trials evaluating the efficacy of CsA for treatment of atopic dermatitis in dogs (10 trials, 672 dogs treated with CsA for a duration of 0.5 to 6 months), Steffan⁶⁶ and coworkers reported that vomiting occurred in 25% of animals and soft stools or diarrhea in 15%. Remaining side effects occurred in less than 3% of the patients. The incidence of infection was not any greater for CsA-treatment groups and may have decreased in the skin and ear.

The commercial form of CsA intended for intravenous use has been associated with anaphylactoid reactions in the dog. The reactions appear to reflect the solubilizing agent. The vehicle of the intravenous preparation is very irritating and must not extravasate.

Monitoring

Monitoring of CsA concentrations can be an important tool to guide effective yet safe CsA regimens. The intent of monitoring should be to establish and maintain the therapeutic range for the patient, and to avoid toxicity or therapeutic failure, including that associated with drug interactions (see Chapter 5). Several points of controversy exist regarding monitoring. Reasons for monitoring in humans include, but are not necessarily limited to, avoiding nephrotoxicity; minimizing the risk of infection; and avoiding therapeutic failure, for which host-versus-graft rejection is the most important. In veterinary medicine monitoring might be implemented for the same reasons, but the need to avoid toxicity might take second stage to the need to assure effective concentrations in the face of marked variability in blood concentrations versus dose relationships. Identifying the intent of monitoring may influence down timing.

Peak, trough, or midinterval samples are variably collected by veterinarians using the author’s laboratory. Yet, the elimination half-life of CsA is short (approximately 5 to 8 hours). Accordingly, the drug generally does not accumulate or only minimally accumulates with multiple dosing. Therefore in most patients, no single sample can accurately predict the concentration throughout the dosing interval to which the patient is exposed. As such, the more critical the maintenance of effective doses throughout a dosing interval, the more helpful both a peak and trough sample might be. Detecting variability in absorption is best accomplished with peak concentrations whereas trough concentrations are influenced by both variability in absorption and elimination. Peak concentrations might be targeted if toxicity is of concern; trough concentrations might be targeted if a minimum effective dose must be maintained. However, trough concentrations run the risk of being nondetectable and failing to predict the marked variability that characterizes CsA absorption particularly with once daily dosing. The least useful sample is that collected mid-interval unless the patient is known to have a very long half-life. Note that if concentrations are to be compared across time (for example, to confirm that a dose increase resulted in higher concentrations), then care must be taken to collect subsequent samples at the same time point in the dosing interval. A difference of two hours can cause concentrations to fluctuate by 25% or more. The farther apart subsequent samples are and the shorter the elimination half-life of CsA in the patient, the less relevant are comparisons between samples collected across time.

Among the important determinants of whether a peak or trough sample is collected is knowing which concentration most accurately predicts response: peak (generally collected at 2 hrs to assure absorption and distribution have been largely completed), trough (just before the next dose, indicating the lowest concentration to which the patient is exposed during an interval) or area under the curve. Timing of sample collection (peak versus trough) is an area of investigation in human medicine. Monitoring traditionally has focused on trough concentrations (discussed later). However, trough (C₀, indicating the lowest concentration) correlates poorly to both graft rejection and acute nephrotoxicity.⁶⁸ Thus peak concentrations (C₂, indicating the highest concentrations anticipated at 2 hours) increasingly are becoming more relevant in humans, not only to predict efficacy but also to avoid toxic effects.⁶⁹ Because toxicity is a lesser concern in animals, trough concentrations might reasonably be preferred to ensure that drug concentrations stay above a minimum effective range throughout the dosing interval. Indeed most animal-based recommendations are based on trough concentrations (see later discussion). However, with the short half-life which characterizes most patients, reaching target concentrations will be easier if a peak sample is consistently collected. This is particularly true if a 24 hour dosing interval is used, for example, when treating atopy. Yet, a half-life of less than 8 hours often results in 24-hour concentrations that are minimally or not detectable. As such, although dose labeled for chronic allergic inflammation (e.g., atopy, asthma), it should not be assumed to be appropriate for autoimmune disorders or graft versus host rejection. For animals in which the CsA half-life is longer than 24 hours, concentrations may not be at steady state. If so, monitoring to establish baseline clearly must be withheld until steady-state concentrations are achieved; monitoring both a peak and trough, however, may be necessary to establish when steady state will occur (see previous discussion).

The impact of drug interactions is another reason for CsA monitoring. If altered clearance is anticipated, elimination half-life is the most likely outcome measure to be impacted that can be monitored. In such instances both a peak and a trough concentration are recommended; if a drug interaction is anticipated (e.g., the addition of ketoconazole), monitoring ideally will occur prior to and approximately 1 week after the inhibiting drug is administered. If a peak and trough concentration cannot be collected, then consistent timing (either a peak or a trough) should be targeted, with a sample before and 1 week after the other drug is changed. Note, however, that if the impact of the drug is to prolong the elimination half-life more than 48 hours, concentrations may not be at steady state at 1 week. The only means by which the duration of time that must elapse before steady state is reached (and thus baseline can be established) is determination of half-life (i.e., both a peak and trough sample). Monitoring should be implemented each time a drug that might alter the disposition of CsA is added or subtracted from the drug armamentarium for the patient.

A second point of concern is the length of time after CsA therapy has begun that monitoring should be implemented. In general, the half-life of CsA is short enough that monitoring can be determined in 3 to 5 days. However, response to CsA may not occur that rapidly. Accordingly, if the goal of monitoring is to establish a therapeutic range in a responding animal, then monitoring is best implemented once a clinical response has been realized This is particularly relevant in the absence of concentration-response data in dogs or cats. The time to response (not resolution) is less clear, but several days to 2 weeks might be reasonable for most targets of therapy. However, some disease may require a longer period for maximum response (e.g., 4 months for perianal fistulae).

Among the limitations in CsA use in dogs and cats is evidence for the the actual concentration to be targeted, which in turn is closely related to timing of sample collection as well as the method used to detect CsA and the sample submitted. Guidelines offered in human medicine have served as targets for veterinary patients,⁶³ although the appropriateness of extrapolation has not been addressed. The impact of the assay reflects the presence of active metabolites (AM1, AM9) which may contribute up to 10 to 20% of activity in humans (more at trough concentrations); the concentration may be even greater if the metabolites accumulate. Several methods are available for measuring CsA concentrations, including HPLC, which generally detects only the parent compound. However, the contribution of active (and potentially toxic) metabolites has led to methods that detect both parent and metabolites.³⁹ The more common methods are immunoassays based on antibodies directed toward the parent (monoclonal) or parent and metabolites (polyclonal). These include radioimmunoassays (RIAs) and polarized immunofluorescence, which detect parent drug and metabolites. Immunoassays that are monoclonal (e.g., FPIA by Abbott TDx or ACMIA by Seimens) are less likely to detect in activemetabolites compared with polyclonal antibody-based assays. Either type of assay (HPLC versus antibody based) is acceptable; the choice might depend on cost or availability. The advantages of antibody-based assays are enhanced quality assurance and rapidity of turnaround time. The cost also should be lower. Each assay appears to be clinically useful. However, the target concentrations will vary with the methodology as well as the timing of the sample (i.e., peak or trough) and the sample collected (e.g., whole blood, plasma, or serum). Antibody-based assays will be accompanied by higher concentrations, with polyclonal concentrations higher than monoclonal. For example, in dogs blood concentrations measured by TDx assay are 1.5 to 1.8 times higher than those measured with HPLC assay.^33,44 However, this increase reflects, in part, detection of active metabolites; as such, monoclonal antibody-based assays that detect the metabolites might be the preferred type. Assays based on HPLC that determine the parent only will have the lowest therapeutic range; HPLC assays that include the metabolites will be higher. A disadvantage of HPLC assays is variability in results among laboratories. Even if similar methods are applied, variability in conditions can result in different concentrations. Thus the importance of using external quality-control programs will be particularly important for laboratories that use HPLC methods.

The sample submitted for CsA quantitation will also influence the concentrations and the therapeutic range. Most CsA in whole blood is located in red blood cells. Accordingly, whole blood is generally the preferred sample. Concentrations based on whole blood will be higher than those measured in plasma or serum. The most important point to be made regarding recommended therapeutic ranges is that they are laboratory specific, varying with sample timing, tissue collection, and assay method. If the laboratory is using an automated system, then the therapeutic ranges of that laboratory will be those established for the instrument. If the laboratory is using HPLC, then the ranges may be specific for that laboratory and only that laboratory. As an example of what might be expected, in humans recommended trough (before next dose) CsA concentrations (ng/mL) are as follows: for HPLC 100 to 300 (whole blood); RIA, monoclonal antibody methodology 150 to 400 (whole blood) or 50 to 125 (plasma or serum); RIA, polyclonal antibody methodology 200 to 800; fluorescent polarized immunoassay 250 to 1000. These numbers are offered as examples of therapeutic ranges. However, therapeutic ranges might also vary with the disease being targeted. A longitudinal replicate study examining differences between laboratories found marked variability within laboratories,⁷⁰ suggesting that intralaboratory variability is a major contributor to overall variability. Because of these reasons, care should be taken not to extrapolate results based on the methodology unless the laboratory fails to do so. However, the most important point to be made regarding therapeutic ranges is that this population statistic is a guide only. Monitoring should be used to determine the patient’s therapeutic range. Once response has been realized, a sample should be collected, the concentration identified, and subsequent samples collected at that same time during the dosing interval.

Monitoring is generally based on 12 hour dosing. The dosing regimen recommended for treatment of atopy⁷¹ should not be assumed to be effective for treatment of immune-mediated diseases. In humans, for which CsA is usually administered by mouth every 12 hours, estimates of drug exposure using the area under the time concentration curve appear to be the most accurate pharmacokinetic measure on which to based dosing in human patients subject to acute rejection episodes. However, the description of area under the curve requires multiple sample collection, which is not only inconvenient but also costly. Two established therapeutic drug-monitoring protocols have been used in humans: the traditional approach, based on trough blood concentrations (C0), and the newer strategy, based on a sample taken 2 hours after oral administration (C2). Because the greatest contributor to interindividual variability in CsA area under the curve in humans is absorption, monitoring that focuses on peak concentrations is evolving as more predictive for CsA. As was noted previously, the risk of graft-versus-host rejection was markedly reduced when CsA dosing was based on peak rather than trough concentrations in humans. However, the use of this parameter as a predictor of CsA exposure is based on a 12-hour dosing interval.⁷² These recommendations are made regardless of the method of CsA detection (mRIA, EMIT, AxSym, CEDIA, or TDx/Architect) (Abbott FPIA). Mehl and coworkers⁴⁵ investigated the relationship between peak and trough plasma drug concentrations and area under the curve in cats receiving CsA. After 14 days of dosing, 2-hour peak concentrations correlated better than the 12-hour trough concentration with area under the curve during that same time period, which suggests that a 2-hour peak sample is the preferred sample for monitoring.⁴⁵ However, this study was based on predicting blood CsA concentrations and not predicting response.

A plethora of literature is dedicated to determining the best peak or trough target concentrations in humans receiving CsA, primarily for transplant patients but also for those with certain chronic allergic inflammatory disorders. For example, 12-hour concentrations, which probably do not accurately predict either rejection or toxicity but are reasonable for maintenance, range from 500 to 800, depending on the author. A review of the literature reveals that the range might also vary during the stage of disease, with initial concentrations for immune-mediated diseases being substantially higher, whereas lower concentrations are acceptable as targets for maintenance once response has been realized. Most recommendations in humans are based on monoclonal antibody assays.⁷²

The consensus among most investigators is that area under the concentration versus time curve is the best predictor of acute or chronic graft rejection and toxicity.⁶⁹ Mahalati and coworkers⁶⁸ demonstrated that the initial area under the concentration-time curve (C₀ and C₄ ; or AUC_0-4) was a useful tool: ranges between 4400 and 5500 ng/mL per hour for the first 4 hours of dosing significantly reduced the risk of acute rejection and nephrotoxicity in humans. This parameter is more useful with newer microemulsion products because CsA disposition is more predictable with these products. For this study samples were collected at 0 (just before the next dose), 1,2, 3, and 4 hours. A similar approach might be considered for animals during the early, critical stages of immune-mediated disease, with sample collection at 0 and 4 hours. However, collection of sufficient samples to describe the curve completely is generally not practical in either human or veterinary patients. For single point prediction, 2-hour posttreatment (C₂) collection has repeatedly been demonstrated to be the most accurate predictor of area under the curve in humans, although even this time point is controversial. However, Einecke and coworkers⁶⁹ demonstrated that C₂ concentrations were not helpful in identifying patients at risk for rejection, but ranges between 500 and 600 ng/mL (whole blood, CEDIA antibody-based assay) were useful to maintain patients for long periods and avoid toxicity. This study suggests that the target range should vary as treatment moves from the induction to the maintenance mode.

Studies correlating CsA concentrations with efficacy are limited in animals. Perhaps the most supportive is with regard to perianal fistulae. Mouatt⁵⁸ found that dogs with perianal fistulae (n = 14) responded more rapidly if trough concentrations exceeded 600 ng/mL, but time to resolution (generally 4 weeks) could not be predicted by concentrations in this small group of dogs.

Use of monitoring for atopic dermatitis is less clear. In a manufacturer-sponsored study, Steffan⁴⁴ and coworkers investigated the relationship between CsA concentrations and response in dogs (n = 97) with atopic dermatitis. Concentrations were measured between 2 and 24 hours of the last dose at 28 days, and this single point concentration was used to predict the time course of CsA, based on a model generated from normal dogs (n = 16). Patients were then grouped into one of five categories of CsA exposure, ranging from very low to very high CsA. Clinical response to therapy was based on scoring of skin lesions (canine atopic dermatitis extent and severity index [CADESI]) and pruritis. Response to therapy (CADESI and pruritis scores) was then compared (not correlated) among groups. The study could not demonstrate a differenc in response among the groups, leading the authors to conclude that concentrations did not correlate with response and monitoring was not indicated. However, this study should not necessary be considered evidence against monitoring in dogs with atopic dermatitis. First, a single time point of CsA was used to predict 24-hour exposure of CsA for clinical patients. Second, the model used to predict CsA was based on normal Beagles, of which only eight had received multiple dosing of CsA. Third, variability in the clinical patients was greater than in normal animals, and the predicted model differed from the observed. The variability in peak CsA, in particular, found in this study is profound, and its impact on therapeutic success should be examined. Variability also occurred in pruritis scores, being equal or greater than the median, which may have impacted the power to detect a significant difference was likely low. Because peak concentrations appear to occur in most animals between 1 and 2 hours, a study that correlates actual peak concentrations with scores might be prudent before the use of CsA monitoring for anticipating response of atopic animals to therapy is set aside. Studies are indicated to determine the best sampling time for dogs receiving CsA. Because concentrations are so low at 24 hours and often undetectable, it is likely that some other sampling period is appropriate for 24-hour dosing intervals.

It is important to emphasize that studies that fail to correlate CsA concentrations with therapeutic response do not prove the lack of or preclude the usefulness of monitoring. In ability to demonstrate a significant difference does not equate to “no treatment” effect. Studies may fail to find a connection because of limitations in study design. In the event that a sufficiently well-designed study (or several studies) should fail to find a correlation, the clinician must remember that the most important reason for monitoring is to establish a therapeutic range for the patient. Once the patient has responded, monitoring might be most useful to maintain that concentration in the patient.

In summary, our laboratory offers the following recommendations for monitoring of patients treated every 12-hours: a 2-hr peak and just before the next dose trough sample is recommended within 3 to 5 days of initiating therapy; the more life threating the target disease, the more important a peak and trough sample may be. For less serious situations, or as treatment shifts from induction to maintenance, a singe 2-hr peak sample or, assuming bid dosing, a single 12 hour trough concentration may be sufficient for establishing and maintaining a target. If therapy is initiated such that CsA disposition might change (whether intentional, such as the addition of ketoconazole, or inadvertent, such as co-treatment with diltiazem or azithromycin or others), a peak and trough sample prior to and 1 week after therapy is initiated is suggested. In situations in which alternatve generic preparations are initiated, a single 2-hr peak concentration before and 3 to 5 days after the switch is recommended. For atopy (24 hr and beyond dosing), a single peak sample should be collected. Likewise, if toxicity is a concern, a single peak sample is indicated. However, in patients with a long half-life, both a peak and trough sample may be necessary to fully assess the risk of toxicity. Monitoring is recommended weekly to biweekly in critical patients, then monthly for the first several months of therapy or until concentrations are stable. For long-term maintenance, the frequency of samply may vary with the stability of the patient but should range from 3 to 6 months. Target concentrations vary with the condition but generally, based on a 12-hr dosing interval using a monoclonal antibody-based assay (i.e., as in the author’s laboratory), a peak concentration of 800 to 1400 ng/mL and a trough concentration of 400 to 600 ng/mL (monoclonal based assay) is recommended for immune mediated diseases. For renal transplantation, trough concentrations of 750 ng/mL are suggested for the first month and 350 to 400 ng/mL, thereafter. For chronic allergic inflammatory disorders, lower concentrations are recommended: 250 ng/mL trough concentrations for chronic inflammatory bowel disorders, and for perianal fistulae, 12 hour trough concentrations at 100 to 600 ng/mL (the higher for induction, the lower for maintenance).

Chronic Allergic Inflammatory Disorders

Bacillus Calmette–Guérin

BCG is a live, attenuated strain of Mycobacterium bovis.⁴ Components of the bacterial cell wall of this organism activate B and primarily T cells; macrophage and neutrophil recruitment in response to lymphokine release results in a granulomatous reaction. The antiviral state induced by BCG appears to be essentially a nonspecific expression of a more or less prolonged stimulation of the immune system. The most likely mechanism for its antiviral activity is direct stimulation of the mononuclear phagocytic system; however, specifically sensitized T lymphocytes that further activate macrophages are generated after a period of time. Stimulated macrophages release colony-stimulating factor, which contributes to macrophage regulation, and IL-1, which promotes lymphocyte proliferation. The antiviral state is thus an indirect benefit of an essentially pathologic, although temporary, situation; the host is characterized by an increased ability to handle viral infections. BCG requires at least 10 days after inoculation to enhance resistance against viral infections. The state of enhanced resistance is, however, long-lasting; repeated administrations (of nonliving product) may lead to a more prolonged and marked effect.¹²⁰

BCG has been used as an adjuvant in combination with tumor vaccines for dogs and cats. Tumors that undergo remission after BCG therapy may do so as a result of the release of tumor-specific antigens during nonspecific tumor lysis. The use of BCG has been studied in combination with a tumor vaccine. In human patients use of BCG as an adjuvant for anticancer therapy has focused primarily on intravesicular administration in bladder cancer.⁴

Muramyl Dipeptide

Muramyl dipeptide is a peptidoglycan and represents the smallest immunologically active component of the Mycobacterium cell wall. Like BCG, it nonspecifically enhances B cell, T cell, and macrophage activities. Because it is rapidly eliminated from the body, it is biologically modified to prolong its actions by incorporation into liposomes or conjugation with glycoproteins. Synthetic production has increased accessibility for clinical use.

Mycobacterium

Mycobacterium vaccae

Ricklin Gutzwiller and coworkers¹²¹ prospectively studied the impact of a single intradermal injection of heat-killed Mycobaterium vaccae on the severity of atopic dermatitis in dogs (n = 62). A double-blinded, placebo-controlled, parallel multicenter study design was used. Dogs were evaluated for 3 months. Skin lesions were scored on the basis of CADESI. The treatment was well tolerated. Treatment was effective in patients with mild or moderate dermatitis but did not affect animals classified as severe. Interestingly, the CADESI scores decreased by 22% in the placebo group (versus 33% in the treatment group).

Propionibacterium acnes

A killed suspension containing P. acnes (formerly C. parvum: Immunoregulin) has been approved for veterinary use (15 μg/kg intravenously biweekly for two to three treatments). Like BCG, it causes complex, nonspecific immunostimulation. Macrophage and cytotoxic T cell activities are enhanced, as is antibody production by both thymus-dependent and thymus-independent antigens. Cell-mediated immunity is also stimulated. It is indicated for the treatment of clinical signs associated with virus-induced and bacteria-induced immunosuppression, such as that caused by FeLV. The efficacy of P. acnes in the treatment of feline infectious peritonitis virus is questionable. One study was unable to demonstrate a difference in survival rate or mean survival time between untreated and treated animals experimentally infected with a high dose of feline infectious peritonitis virus. Studies investigating the antitumor effects of Propionibacterium acnes in dogs do not support its efficacy against early neoplasia, although it may be more effective in advanced disease when used in combination with other drugs.²⁵ Cats infected with FeLV and afflicted with various non-neoplastic diseases showed some signs of response to treatment with P. acnes (Immunoregulin), although no cat reverted to an FeLV-negative status.²³ Studies regarding the efficacy of P. acnes in FeLV-induced tumors apparently have not been done.²⁵

Staphylococcal Protein A

Staphylococcal protein A (SpA) is a cell wall polypeptide of S. aureus Cowan I (SAC) that binds rapidly and with great affinity to immune complexes. Other regions of the molecule initiate T lymphocyte and B lymphocyte proliferation as well as secretion of soluble lymphocytic products such as IFN. Circulating immune complexes have been incriminated as “blocking factors” that aid in a tumor’s escape from immunologic control. Therapeutic trials of the efficacy of SpA have been based on the hypothesis that removal of specific or nonspecific immunosuppressive molecules such as blocking factors will enhance host immune response to the tumor. Other regions of the SpA molecule initiate T lymphocyte and B lymphocyte proliferation and secretion of soluble lymphocyte products such as IFN. Treatment with SpA is achieved by extracorporeal perfusion of host plasma over whole SAC organisms or through filters containing purified SpA. Studies have shown that SpA or SAC treatment reduces tumor size, decreases levels of circulating immune complexes, and induces the appearance of cytotoxic antibodies directed toward neoplastic cells. A study in which SpA was used to treat FeLV-infected cats found that 50% of cats with FeLV-associated disease improved, and 33% of cats afflicted with malignant disease responded with reductions in tumor size as well as in bone marrow and peripheral blood neoplastic cells.^{23,25,122,123} Viremia was cleared in 28% of treated cats. Circulation of a g-like IFN was demonstrated in some cats that responded to SpA and was followed by the appearance and rising titer of complement-dependent cytotoxic antibody. The antibody reacted with FeLV-infected cells and was specific for the major viral envelope glycoprotein gp70. The ability of cats to persistently remain FeLV negative appeared to correlate with the magnitude of FeLV antibody titer.²⁵

Mixed Bacterial Vaccines

Mixed bacterial vaccines composed of Streptococcus pyogenes and Serratia marcescens have been studied in cats with malignant mammary tumors. Although not statistically significant, survival time was greater (875 days) when surgery was combined with mixed bacterial vaccines versus surgery alone (450 days).

Cytokines

Biological response modifiers of natural original also are discussed in Chapter 32. A number of cytokines produced by leukocytes and related cells actively regulate the immune system. Recombinant DNA technology has led to widespread production and greater application of drugs that modulate cytokines or their receptors or transcription factors in the treatment of immunologically based disease. The cytokines most likely to be used for immunostimulatory effects include the IFNs and selected interleukins.

Passive Immunotherapy: Antibodies

Passive immunotherapy with monoclonal or polyclonal antibodies has been investigated for both the diagnosis and therapy of cancers. Antibodies are directed toward surface antigens expressed by tumor cells. Specificity of antibodies (as long as Igs are derived from the species intended to be treated) should limit host toxicity to those cells expressing the antigen (i.e., tumor cells), thus avoiding systemic toxic effects. The antibodies themselves may be directly toxic to the cell or induce complement-dependent cytotoxicity. In addition, antibodies can be conjugated to drugs, toxins, or radioisotopes toxic to tumor cells, thus limiting the specificity of a variety of pharmaceuticals for tumor cells.

Igs available in plasma obtained from donors generally contain detectable antibodies against bacterial, fungal, and viral pathogens.⁴ Passive transfer of resistance to the immunodeficient patient can be accomplished with intramuscular or intravenous administration. In human patients the half-life of transferred Igs is approximately 3 weeks.⁴ Indications for human patients have included congenital or acquired states of immunodeficiency, selected hematologic disorders such as autoimmune hemolytic anemia, infectious viral diseases, and selected neoplastic diseases such as chronic lymphocytic leukemia and multiple myeloma. Potential adverse reactions include anaphylaxis and transfer of infectious organisms.⁴

Studies investigating the efficacy of passive immunotherapy on feline lymphosarcoma have thus far been limited to the use of polyclonal FeLV-neutralizing antibodies, feline oncornavirus-associated cell membrane antigen (FOCMA) antibodies, or both in cats afflicted with lymphosarcoma and leukemia. In one study five of seven cats treated with both antibodies and six of eight cats treated with FOCMA antibodies alone underwent partial or complete regression of their disease.²⁵ Additional studies by Cotter and coworkers¹²⁴ suggest that polyclonal anti-FOCMA therapy combined with previously established chemotherapeutic regimens may improve remission time. Monoclonal antibodies have been used to treat canine lymphosarcoma.

Human intravenous immunoglobulin (hIVIG) has been studied in dogs using in vitro (lymphocytes and monocytes) and in vivo (spontaneous immune-mediated diseases) methods. Prepared from plasma of healthy donors, it contains polyspecific IgG and is intended to treat primary immunodeficiencies. In vitro studies¹²⁵ have shown hIVIG to bind to canine lymphocytes and monocytes and to inhibit through Fc-mediated binding monocyte phagocytosis, including that of antibody-coated red blood cells. It is administered as an intravenous infusion (0.5 to 1.5 g/kg) over 6 to 12 hours, and animals must be closely monitored for signs of anaphylaxis or other adverse reactions (e.g., thromboembolism). Clinical evidence of efficacy exists for treatment of IMHA,^126-128 with treated dogs developing an increased hemoglobin and hematocrit concentration up to 4 weeks after therapy. However, 50% of treated animals developed thrombocytopenia and five of seven dogs that died had evidence of thromboembolism. Only 3 of 10 dogs were alive at 12 months, suggesting that long-term survival was not improved. The combination of IgG with IFN α resulted in greater improvement in human patients with hepatitis C.¹²⁹

Abetimus is a synthetic molecule consisting of four double-stranded oligodeoxyribonucleotides attached to polyethylene glycol (PEG). The nonimmunogenic PEG serves as the carrier. Abetimus induces tolerance to B cells by cross-linking surface antibodies directed toward double-stranded DNA (dsDNA). These antibodies appear to be associated with nephritis associated with systemic lupus erythematosus in humans.¹³⁰ In a preapproval clinical trial, the number of renal “flares” in patients receiving the drug was less than half of that encountered in patients not receiving the drug. The response was so dramatic that the trial was discontinued before its completion, and the FDA granted orphan drug status in 2000.¹³⁰

Allergen-Specific Immunotherpy

Allergen-specific immunotherapy (ASIT) involves the administration of gradually increasing amounts of an allergen extract to patients allergic to the allergen to resolve symptoms associated with subsequent exposure to the allergen.¹³¹ In their review Colombo and coworkers¹³¹ note that when success is defined as a 50% (or greater) increase in the improvement of clinical signs after 4 months or more of therapy, ASIT is successful in 50% to 100% of cases of canine atopic dermatitis. They prospectively evaluated the effect on canine atopic dermatitis of low (1⁄10 the standard) dose induction followed by maintenance ASIT therapy (n = 27) to standard therapy (n = 13). Both groups responded, and neither group emerged as responding better at 9 months. Secondary infections were treated, and use of glucocorticoids was allowed to control severe clinical signs, except during the last 3 months of the study.

Miscellaneous Blood Components

Selective destruction of malignant lymphocytes occurs in several species in response to infusion of blood or blood components.

Levamisole

Levamisole, a phenylimidazothiazide anthelmintic, has been the subject of intense and controversial research as a biological response modifier.^23,120 It is difficult to summarize the experimental and clinical data regarding the immunomodulating capabilities of levamisole. Levamisole has been regarded by some as a chemical agent capable of mimicking hormonal regulation of the immune system. It appears to stimulate recruitment and function of macrophages and T cells but only within a narrow range of doses and duration of administration in either the normal or immunoincompetent patient. Levamisole appears to alter cyclic nucleotide phosphodiesterases, decreasing cyclic guanosine monophosphate degradation and increasing cyclic adenosine monophosphate degradation. Elevated cyclic guanosine monophosphate in lymphocytes enhances proliferation and secretory responses. Chemotaxis, phagocytosis, lymphokine synthesis, and the ratio of helper to suppressor T cells are increased. Levamisole does not appear to have any effect in immunocompetent animals. The modulatory effects of levamisole range from enhancement to inhibition with much strain, sex, age, and antigen variability. T cell stimulation may be mediated by a soluble serum factor. Experimental studies generally have not supported the benefits of levamisole as have clinical studies. Levamisole (2 to 15 mg/kg) was frequently tested prophylactically in experimental trials, however, as opposed to therapeutically (i.e., in infected patients) in clinical trials, suggesting that potential benefits of levamisole result from restoration of immunocompetence after virally induced immunosuppression. Indications for levamisole in human medicine include Hodgkin’s disease and rheumatoid arthritis and as an adjuvant chemotherapy of colorectal cancer.⁴ The use of levamisole for treatment of cancer in animals is not supported. In two studies involving more than 130 cats with malignant mammary tumors, levamisole (5 mg/kg orally on 3 alternate days per week) did not increase survival time or decrease recurrence rate.¹³⁹ Levamisole causes adverse reactions typical of excessive cholinergic stimulation.

Cimetidine

Histamine exerts immunomodulatory effects, including suppression of cytotoxic T lymphocytes, downregulation of cytokines, and activation of suppressor T lymphocytes.¹⁴⁰ Cimetidine is a histamine (H₂) receptor antagonist that experimentally enhances a variety of immunologic functions. Suppressor T lymphocytes possess H₂ receptors, which, when blocked, result in potentiation of cell-mediated immunity. Although more studies are indicated, cimetidine may be useful in a variety of conditions associated with immunosuppression. Because it selectively inhibits suppressor function, however, cimetidine also may prove deleterious to patients with autoimmune disorders.

The anticancer effects of cimetidine have been studied in selected human cancers. The H₂ receptor blockers have been studied for their effects on gastric cancer cells. Cimetidine, but not famotidine, exhibits antiproliferative effects on gastric cancer cells. Raniditine showed some inhibitory effects.¹⁴⁰ Cimetidine also appears to inhibit the growth of colorectal carcinoma;¹⁴¹ lymphocyte infiltration increases in the cancers and is associated with an improved survival rate in patients receiving the drug.

ABPP (Bropirimine)

ABPP (2-amino-5-bromo-6-phenyl-4[³H]-pyrimidinone), approved for use in humans, is a potent inducer of IFN in several species. Hamilton and coworkers¹⁴² characterized the kinetics of ABPP and induced IFN in several species. They found that serum levels of IFN after treatment correlated well with serum concentrations of ABPP in the cat. ABPP was lethal in three of eight cats, however, at doses required to achieve minimal detectable levels of IFN. The authors postulated that the toxicity resulted from conversion of ABPP to phenolic derivatives that the cat excretes inefficiently.

Isoniplex

Isoniplex is an antiviral drug (see Chapter 10) that may also be used as an immunopotentiator in viral diseases.^23,120 Isoniplex enhances immune responses, including promotion of mitogen-stimulated T-lymphocyte proliferation and augmentation of antibody production and delayed-type hypersensitivity. The suggested mechanism of immunopotentiation by isoniplex begins with penetration of lymphocytes and suppression of viral RNA synthesis. It appears to support lymphocyte function by promoting RNA synthesis and translational ability. Further investigations regarding the use of isoniplex in viral infection should be anticipated because its efficacy results from an ideal combination of antiviral activity and immunopotentiation.

Promodulin

Promodulin is an experimental immunomodulating agent that has been subjected to clinical therapeutic trials for the treatment of cats concurrently infected with FeLV and feline infectious peritonitis.²³ Cats were treated with 50 mg/kg (up to 200 mg maximum dose) intravenously once daily for 5 consecutive days. Although promodulin induced rapid remission of clinical signs associated with feline infectious peritonitis, (e.g., anorexia, fever, and serosal effusions), it did not appear to be effective in the treatment of concurrent FeLV infections. Cats that responded did so within 2 weeks of the final injection; the duration of clinical remission appeared to vary between 1 and 3 months. Clinical signs after exacerbation of disease did not respond to a second treatment regimen. Promodulin also was not effective in treating FeLV-induced solid tumors.

Imidazoquinolines

Imiquimod and resiquimod (R-848) are members of the family imidazoquinolines. These drugs have proven antiviral and antitumor effects in a variety of animal models. Their immune actions appear to reflect stimulation of several arms of both innate and adaptive immunity, ultimately resulting in an indirect T_h-1 dominant response. Their ability to enhance cutaneous-adaptive responses and innate immunologic responses are key to their usefulness. Resiquimod is 10- to 100-fold more potent than imiquimod and is also capable of stimulating granulocyte and macrophage colony-stimulating factors and other mediators. Antigen processing also is improved. Indications include cutaneous viral infections and potentially nonmelanoma skin cancers, with the latter having potential therapeutic applications in animals.¹⁴³

Anaphylaxis and Anaphylactoid Reactions

Clinical signs of systemic anaphylaxis include nausea, vomiting, diarrhea, pale mucous membranes, coolness to peripheral extremities, tachycardia, and tachypnea. Localized “anaphylaxis” results in clinical signs referable to the site of localized mast cell degranulation. Examples include angioneurotic edema resulting in swelling of lips, eyelids, and conjuctiva and urticarial lesions (or hives). Treatment is oriented toward preventing further mast cell degranulation, blocking the interaction between histamine (or other mediator) and tissue receptors, and antagonizing the physiologic response to mediators. Drugs that antagonize physiologic response also tend to further decrease mast cell degranulation. The goals of therapy for systemic anaphylaxis include cardiovascular and ventilatory support. Epinephrine is indicated to antagonize bronchoconstriction and provide cardiovascular support. Glucocorticoids (prednisolone sodium succinate) facilitate adrenergic receptor responses and decrease further mast cell mediator release. Histamine 1 (H₁) receptor antagonists (e.g., diphenhydramine) are of benefit only in preventing interaction of histamine and its receptors, not in preventing further mast cell degranulation. Therapy may be more effective if administered in anticipation of mast cell degranulation, although this is somewhat controversial. Perioperative anaphylaxis, including drugs, risk factors, and therapies in humans, has been recently reviewed.¹⁵³

Immune-Mediated Hemolytic Anemia

IMHA occurs as a result of increased red blood cell destruction mediated by the presence of an antibody on the membrane surface. The antigen to which the antibody binds is either of the red blood cell membrane or an exogenous antigen (e.g., drug or microbe) that has adhered to the surface. Igs associated with IMHA in dogs generally are IgG or, less commonly, both IgG and IgM. In cats IgM tends to be more common, with IgG alone causing IMHA in approximately 25% of cases. Complement activation is more likely with IgM-mediated IMHA. Antibody adherence and complement activation that are insufficient to cause erythrocyte lysis will result in damage and subsequent erythrophagocytosis of the deformed red blood cell (e.g., spherocyte). Intravascular hemolysis occurs when complement activation is extensive, leading to erythrocyte lysis. Released hemoglobin binds to serum haptoglobin, preventing glomerular filtration. If haptoglobin becomes saturated, however, free hemoglobin can be filtered. Renal toxicity can accompany intravascular IMHA as a result of antigen–antibody deposition or reaction in the basement membrane; free hemoglobin may also contribute to nephrotoxicity. Direct red blood cell agglutination or intravascular hemolysis increases the risk of thromboembolic disease.

Medical treatment of IMHA focuses on reducing phagocytosis of damaged or antibody-coated erythrocytes by reducing or blocking receptors on phagocytic cells, reducing or preventing the formation of more antibodies, and providing supportive therapy for complications associated with IMHA. Any likely inciting (exogenous) antigen (e.g., drug, microbe) should be removed. Treatment of IMHA is confounded by low survival rates (which range from 10% to 50% discharge survival, with continued patient loss after discharge) and the lack of scientifically based evidence supporting preferred therapies. Several of the clinical trials that address various therapeutic regimens unfortunately are biased in population selection, generally focusing on the worse cases and thus complicating extrapolation of results to less severe cases. Immunosuppressive therapy with glucocorticoids is the cornerstone of therapy; use of immunosuppressants for immune-mediated disorders is reviewed in Chapter 30. Differences of opinion exists regarding the initial route of administration of glucocorticoids, with oral proponents observing that response to glucocorticoids requires 5 to 7 days regardless of route. Although this observation may have some merit (many mechanisms of glucocorticoid require nuclear transcription and protein synthesis; time must elapse before inflammatory cell numbers and function are reduced), many mechanisms (e.g, non-genomic) of glucocorticoid action may occur immediately. Indeed, the importance of glucocorticoid supplementation in shock patients offers support for administration designed to cause immediate response (see Chapter 30). For rapid response dexamethasone (0.1 to 0.2 mg/kg intravenously) or methylprednisolone (11 mg/kg daily for up to 3 days) every 12 to 24 hours is administered initially, followed by oral prednisolone (1 mg/kg every 12 hours) as the animal responds. Choice of prednisone versus prednisolone is addressed in Chapter 30; the latter is strongly encouraged in dogs with life-threatening conditions, and the former should not be used in cats. Further reduction of red blood cell destruction may require administration of CsA, leflunomide or MMF, or their combination; continued failure may require cyclophosphamide or (dogs only) azathioprine. Combination therapy might be considered initially in patients with severe disease (see Chapter 30). Leflunomide doses as high as 8 mg/kg were necessary to prevent renal transplant rejection in dogs. ^104a Safety of anticancer drugs can be increased by dosing based on body surface area. Cyclophosphamide can be given as a single intravenous bolus (100 to 250 mg/m²) in patients suffering from intravascular lysis or direct autoagglutination.¹⁰⁸ This regimen also is particularly useful for patients that require a blood transfusion. However, efficacy of cyclophosphamide may not be realized until antibodies decline as a result of normal catabolism (generally 1 to 2 weeks).¹¹ Oral administration (50 mg/m² every 48 hours) is indicated in dogs that do not respond to glucocorticoids or might be considered as part of initial therapy, particularly in severe cases. Using a randomized prospective design, Mason and coworkers¹⁵⁴ investigated the impact of cyclophosphamide (50 mg/m²) when combined with prednisone (1 to 2 mg/kg orally every 12 hours; n = 8) compared with prednisone alone (1 to 2 mg/kg orally every 12 hours; n = 10) for treatment of severe (based on clinical signs, less than 1 week in duration and packed cell volume less than 20%) idiopathic IMHA in dogs. The mortality rate did not differ between groups, but the power of the study was limited. Reticulocytosis appeared, and spherocytosis resolved more rapidly in the prednisone-only–treated group (within 1 week), leading the investigators to conclude that the addition of cyclophosphamide offered no advantage to prednisolone alone. Long-term cyclophosphamide will increase the risk of thrombocytopenia and neutropenia.¹¹

Human gammaglobulin (0.5 to 1.5 g/kg intravenously over 6 to 12 hours) may prove useful for dogs that fail to respond to therapy.^127,128 Cost may, however, be prohibitive, and use may be limited by development of thrombocytopenia or thrombosis (see previous discussion). CsA may be of benefit in controlling the sequelae of IMHA that reflect tissue (and thus T cell–mediated) damage.¹¹ Blood transfusions are not recommended for patients with IMHA. Dogs with IMHA are predisposed to destruction of transfused red blood cells. Blood substitutes (oxyhemoglobin) may offer a viable alternative to blood transfusion. Although splenectomy should be considered as a surgical adjunct to medical management, removal of the spleen may also result in removal of an important site of extramedullary hematopoiesis. As with glucocorticoids, danazol (5 to 10 mg/kg orally every 12 hours) may block Fc receptors on phagocytic cells, reducing red blood cell destruction.¹¹¹ However, response may be slow, rendering danazolol lower on the list.

Response to therapy is based on daily hematocrit or platelet counts (or both); initial therapy should be continued or, as needed, improved until the packed cell volume is 15% or more in cats and 20% in dogs or the platelet count is 100, 000/μL or more. Drug therapy should be gradually tapered to avoid relapse or rebound; for example, prednisolone and azathioprine tapered to 0.5 to 1 mg/kg every second day is a reasonable target, with monitoring every 2 weeks as a basis of response. Therapy should be continued for another 3 months or so, with gradual tapering off attempted in animals that have been in remission for 3 to 6 months. For nonresponders, supportive therapy may include transfusions, which are more likely to be effective in patients whose disease reflects bone marrow rather than peripheral cellular targets.

Other supportive therapy should be considered. Anemia and reduced oxygen delivery to the gastrointestinal tract may predispose the patient to gastrointestinal induced ulceration. The importance of cyclooxygenase-2 in gastrointestinal healing warrants consideration of gastroprotectants, particularly in the face of glucocorticoid therapy. As such, antisecretory drugs (e.g., H₂-receptor antagonists, omeprazole) and gastroprotectants (e.g., sucralfate) should be considered as long as concerns regarding drug interactions are addressed. The use of heparin, including fractionated products, is controversial but might be considered (Chapter 15).

Immune-Mediated Thrombocytopenia

Immune-mediated thrombocytopenia is a syndrome that occurs more commonly in dogs than cats and in males than females. It can occur in concert with other immune-mediated disorders, including IMHA.¹¹ Thrombocyte numbers can decline as a result of destruction (i.e., antibody/complement-mediated phagocytosis) or, less commonly, decreased formation of mature thrombocytes as a result of antibody/complement-mediated destruction of megakaryocytes.¹¹ As with IMHA, antibody can be directed to an endogenous or exogenous antigen adhered to the platelet or megakaryocyte. The primary clinical signs, which include and reflect inappropriate bleeding, generally do not occur until thrombocyte numbers have dropped below 30,000/μL. Bleeding is more likely with a rapid, as opposed to a gradual, decline.

Medical treatment focuses on prevention of bleeding, decreased destruction of thrombocytes, and restoration of thrombocyte numbers (see also Chapters 15 and 30). Any likely inciting (exogenous) antigen (e.g., drug, microbe) should be removed. Glucocorticoids (dexamethasone, methylprednisolone, or prednisolone) are often the first choice of therapy (see earlier discussion of IMHA) and, in general, cause thrombocyte counts to normalize within 1 week. Use of cyclosporine and leflunoamide should be considered as suggested for other immune-mediated disease. Danazol also can be administered to reduce phagocytosis.^112,113 Vincristine (0.75 mg/m² intravenously) can be administered if platelet numbers fail to increase to sufficient numbers. Once platelet numbers increase, vincristine should be discontinued and glucocorticoids continued for several more weeks.¹¹ Vincristine may also be given after incubation with platelet-rich plasma in cases refractory to glucocorticoids, danazol, and vincristine. Presumably, incubation allows vincristine to bind to platelets. Subsequent phagocytosis destroys the phagocytic cell, causing an overall reduction in platelet destruction.¹⁵⁵ If successful, the treatment may need to be repeated as new macrophages are generated. Platelet-rich plasma also can be administered in an attempt to restore platelet numbers to a concentration that is not life threatening (>50,000/μL). The likelihood of relapse of immune-mediated thrombocytopenia also may be reduced after infusion of platelet-rich plasma.¹¹ The use of vinblastine might be considered in relapsing patients (see discussion of cytotoxic drugs) on the basis of its effectiveness in human patients. Splenectomy is a surgical alternative or adjunct therapy that should be considered in refractory cases. Because of the risk of relapse, patients should be monitored periodically. Surgical neutering, particularly of females, may be indicated once platelet numbers have normalized.

Immune-Mediated Neutropenia

As with other immune-mediated hematopoietic diseases, glucocorticoids are indicated for neutropenia.¹⁵⁶ Treatment can continue as described for IMHA.

The use of recombinant bone marrow growth factors (see Chapter 15 Chapter 32) to increase bone marrow production of deficient cells is probably not wise unless the factor is derived from the species to be treated. Even in those situations, studies should confirm a lack of immune-mediated reactions when used in patients affected with an immune-mediated disorder.

Dermatologic Disorders

A number of immune-mediated skin diseases reflect a type II hypersensitivity. Included are pemphigus (foliaceus, erythematosus, vulgaris, vegetans), bullous pemphigoid, and dermatomyositis.

Systemic Lupus Erythematosus

The deposition of circulating autoantibodies or autoantibody–antigen complexes in the endothelium, particularly that of the glomerulus, appears to initiate complement-mediated inflammation. The inflammatory site is infiltrated by immune cells. In the glomerulus response includes proliferation of capillary cells, thickening of the basement membrane, and scarring. Other vascular beds affected include the skin, serous membranes, synovial tissues, and cutaneous and visceral blood vessels.^114,115 Soluble immune complexes are more problematic than large immune complexes, which precipitate and are rapidly phagocytized. Soluble complexes are able to penetrate deep into vascular endothelial channels, activate complement, and stimulate an inflammatory response. In humans intravenous cyclophosphamide has been established as the treatment of choice for lupus-induced nephritis. Bolus cyclophosphamide has also, however, proved effective for lupus affecting other body systems, including the CNS, lungs, and arteries. Azathioprine and weekly methotrexate are effective for treating human lupus that does not involve major organs, such as rashes, serositis, and arthritis, or in combination with glucocorticoids to reduce the glucocorticoid dose. CsA apparently has not been studied for treatment of systemic lupus erythematosus, although clinical response has been reported.^114,115

Because systemic lupus erythematosus can be polysystemic in its presentation, the sequelae of immune complex deposition associated with systemic lupus erythematosus can affect a number of body systems, resulting in the need for medical management of secondary disease. Examples include but are not limited to glomerulonephritis, arthritis, and vasculitis. The reader is referred to the chapters that address these specific body systems for information on management of the sequelae of inflammatory disease. Treatment for the immune-mediated aspect of the disease is the same as for pemphigus skin disorders. Use of cyclosporine and leflunoamide should be considered as suggested for other immune-mediated disease.

Cutaneous Discoid Lupus Erythematosus

Considered a mild form of systemic lupus erythematosus, cutaneous discoid lupus erythematosus is not accompanied by systemic involvement. The most common form presents as a nasal dermatitis; skin surrounding the eyes, pinnae, lips, and feet may also be involved. Treatment includes immunosuppressive doses of glucocorticoids, as described for other immune-mediated diseases, although a lower initial dose may be effective (1 mg/kg orally twice daily). Vitamin E (400 IU orally 2 hours before or after a meal every 12 hours; acetate or succinate) may be effective as the sole agent; however, a 30- to 60-day lag time to efficacy mandates that initial therapy include glucocorticoids. Topical glucocorticoids may be effective in mild cases. Minimizing exposure to the sun, including use of topical sunscreen, or other ultraviolet light also will be helpful, particularly in animals with depigmentation. Niacin or tetracycline (5 to 12 mg/kg orally every 8 hours) was reportedly effective in 70% of cases in one study. Use of cyclosporine and leflunoamide should be considered as suggested for other immune-mediated disease; tacrolimus also might be considered.

Rheumatoid Arthritis and Other Arthritides

The rheumatoid factor is an antibody that reacts with IgG that has bound to antigen and subsequently undergone a conformation change. The reason for selectivity in joints is not understood. In humans drugs used to treat rheumatoid arthritis include gold compounds or penicillamine and cytotoxic drugs, including azathioprine, cyclophosphamide, and methotrexate. Since the late 1980s, low-dose weekly methotrexate has become the preferred medication.^114,115 Methotrexate has proved more rapid in onset. Because it is safer than traditional cytotoxic drugs, treatment generally can progress for a longer period of time. Because functional disabilities and progress of the disease occur rapidly in the first years of disease in humans, cytotoxic drugs are begun early. Drug combinations have been advocated because of the possibility of synergistic effects. Examples include methotrexate with sulfasalazine, CsA, or biological agents. Treatment in animals has not been as well investigated and focuses on control of inflammation with glucocorticoids and, if necessary, aspirin. The use of glucocorticoids is supported by the rapid clinical improvement and decrease in IL-6 activity documented in dogs with juvenile polyarthritis syndrome.¹⁶² The use of other immunosuppressive drugs in animals (azathioprine, cyclophosphamide) has been reserved for severe cases. On the basis of findings in humans, however, a more aggressive approach may be warranted. Disease-modifying agents (e.g., glucosamine and chondroitin sulfates) should be used to support cartilage repair.

Feline chronic progressive polyarthritis is associated with feline leukemia virus and feline syncytium-forming virus and most commonly presents as osteopenia and periosteal bone proliferation around affected joints. Less commonly, joints are characterized by subchondral marginal erosions similar to those of rheumatoid arthritis. Rheumatoid factor cannot be identified, however. Treatment includes immunosuppressive doses of corticosteroids (1 to 3 mg/kg orally every 12 hours). Nonresponders may require treatment with chlorambucil, cyclophosphamide, or azathioprine. As previously suggested, disease-modifying agents may prove beneficial.

In a retrospective study of dogs (n = 39) with immune-mediated polyarthritis, 56% of dogs were cured after drug therapy. Response to prednisolone alone was 50% (overall 33%), although response to prednisolone (1 to 2 mg/kg every 24 hours or divided every 12 hours) by itself or combined with another drug was 81%. Duration of therapy was generally 4 to 16 weeks, although continuous medication was necessary in 10% to 18%. Response of animals to leflunomide was previously discussed.^104a Drugs combined with prednisolone to which animals responded included antimicrobials and immunosuppressive drugs such as cyclophosphamide or azathioprine.¹⁶¹ Treatment with levamisole or CsA as sole therapy generally was not successful.

Allergic Contact Dermatitis

Allergic contact dermatitis is the most common type IV hypersensitivity recognized in small animals, being responsible for up to 10% of dermatologic cases.¹¹ The syndrome is initiated when the skin comes in contact with the inciting antigen or chemical. Actual chemicals that cause contact dermatitis are not known, but it is likely that the chemical acts as a hapten that subsequently covalently bonds to a protein. The location of the protein is not clear but is probably associated with the class II molecule of an APC, which in the skin is the Langerhans cell. Sensitization generally requires 4 to 10 days; subsequent exposure to the chemical results in a marked T cell–mediated response.¹¹ Treatment is best implemented by removal of the inciting antigen and short-term (7-day) administration of prednisolone (0.5 to 1 mg/kg every 12 to 24 hours).

Inflammatory Myopathies

In humans polymyositis appears to reflect cell-mediated, antigen-specific cytotoxicity, with azathioprine being the only cytotoxic drug to be of benefit in controlled studies. Low-dose methotrexate or azathioprine with high-dose glucocorticoid therapy has become the standard therapy. Combinations of methotrexate and either cyclophosphamide or CsA may be effective and are being studied.^114,115

Inflammatory Bowel Disease

Inflammatory bowel disease also is discussed in Chapter 19. The use of immune-modifying therapy for human patients with IBD became popular only in the 1990s.^{114,115,164,165} Controlled clinical trials in humans have focused on azathioprine, 6-mercaptopurine, CsA, and methotrexate. Azathioprine or 6-mercaptopurine has been effective for treatment of Crohn’s disease, although efficacy depends on duration of therapy; at least 13 and more often 17 weeks of therapy is required before the drugs reach their full effects.

Intravenous administration of azothioprine may decrease the time to response. CsA has been studied at low doses (<1 mg/kg per day) or high doses (>5 mg/kg per day) to minimize the risk of nephrotoxicity (less of a concern in veterinary patients), but this has proved of little benefit in Crohn’s disease. It has been more effective for treatment of ulcerative colitis at high doses (8 to 10 mg/kg per day), although only a few patients have been studied. CsA concentrations in responding patients were above 250 ng/mL. Methotrexate appears to be useful for both induction of remission and steroid sparing in patients with Crohn’s disease and ulcerative colitis. Combinations of drugs also have been studied. Azathioprine and 6-mercaptopurine should not be used in combination with methotrexate because of the increased risk of toxicity. CsA and methotrexate have been used in human patients with IBD with some success. Drugs that target leukotrienes should be considered. Pentoxyfylline for treatment of human IBD is discussed in Chapter 19.